Akt activates NOS3 and separately restores barrier integrity in H2O2-stressed human cardiac microvascular endothelium.
The integrity of microvascular endothelium is an important regulator of myocardial contractility. Microvascular barrier integrity could be altered by increased reactive oxygen species (ROS) stress seen within minutes after cardiac arrest resuscitation. Akt and its downstream target nitric oxide (NO) synthase (NOS)3 can protect barrier integrity during ROS stress, but little work has studied these oxidant stress responses in human cardiac microvascular endothelial cells (HCMVEC). We, therefore, studied how ROS affects barrier function and NO generation via Akt and its downstream target NOS3 in HCMVEC. HCMVEC exposed to 500 microM H2O2 had increased Akt phosphorylation within 10 min at both Ser-473 and Thr-308 sites, an effect blocked by the phosphatidylinositol 3-kinase inhibitor LY-294002. H2O2 also induced NO generation that was associated with NOS3 Ser-1177 site phosphorylation and Thr-495 dephosphorylation, with Ser-1177 effects attenuated by LY-294002 and an Akt inhibitor, Akt/PKB signaling inhibitor-2 (API-2). H2O2 induced significant barrier disruption in HCMVEC within minutes, but recovery started within 30 min and normalized over hours. The NOS inhibitor Nomega-nitro-L-arginine methyl ester (200 microM) blocked NO generation but had no effect on H2O2-induced barrier permeability or the recovery of barrier integrity. By contrast, the Akt inhibitor API-2 abrogated HCMVEC barrier restoration. These results suggest that oxidant stress in HCMVEC activates NOS3 via Akt. NOS3/NO are not involved in the regulation of H2O2-affected barrier function in HCMVEC. Independent of NOS3 regulation, Akt proves to be critical for the restoration of barrier integrity in HCMVEC.
Dossumbekova
Anar
A
Department of Medicine, MC5068, Section of Emergency Medicine, Emergency Resuscitation Ctr., The University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA.
Berdyshev
Evgeny V
EV
Gorshkova
Irina
I
Shao
Zuohui
Z
Li
Changqing
C
Long
Phillip
P
Joshi
Atul
A
Natarajan
Viswanathan
V
Vanden Hoek
Terry L
TL
D002199:Capillary Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D003331:Coronary Vessels / Q000187:drug effects* / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D055806:Microvessels / Q000187:drug effects* / Q000201:enzymology; D009025:Morpholines / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009705:Nucleosides / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011724:Pyridazines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors
D002867:Chromones; D006861:Hydrogen Peroxide; D009025:Morpholines; D019331:NG-Nitroarginine Methyl Ester; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D009705:Nucleosides; D016877:Oxidants; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D011724:Pyridazines; D017382:Reactive Oxygen Species; C488733:API-2 nucleoside; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C496314:NOS3 protein, human